Oxalate Oxidase for In Situ H2O2‐Generation in Unspecific Peroxygenase‐Catalysed Drug Oxyfunctionalisations

Abstract H2O2‐driven enzymes are of great interest for industrial biotransformations. Herein, we show for the first time that oxalate oxidase (OXO) is an efficient in situ source of H2O2 for one of these biocatalysts, which is known as unspecific peroxygenase (UPO). OXO is reasonably robust, produces only CO2 as a by‐product and uses oxalate as a cheap sacrificial electron donor. UPO has significant potential as an industrial catalyst for selective C−H oxyfunctionalisations, as we confirm herein by testing a diverse drug panel using miniaturised high‐throughput assays and mass spectrometry. 33 out of 64 drugs were converted in 5 μL‐scale reactions by the UPO with OXO (conversion >70 % for 11 drugs). Furthermore, oxidation of the drug tolmetin was achieved on a 50 mg scale (TONUPO 25 664) with 84 % yield, which was further improved via enzyme immobilization. This one‐pot approach ensures adequate H2O2 levels, enabling rapid access to industrially relevant molecules that are difficult to obtain by other routes.


S2.4. Scheme
Scheme S1. Catalytic cycle of UPOs S2.5. Tables   Table S1. Comparation of H2O2-generation systems for ethylbenzene hydroxylation catalysed by AaeUPO Table S2. Drug stocks prepared for 5 µL-scale reactions (Fig. S18-19) S3. References S4. Author Contributions S1. Experimental Procedures S1.1. Preparation of DNA constructs Electra reagents kit (ATUM, including type IIS restriction enzyme SapI) was used for cloning in either pD902 or pD912 vectors (ATUM). Agrocybe aegerita unspecific peroxygenase (AaeUPO) gene (GenBank: FM872458.1) containing nine mutations (PaDa-I) [1] was kindly provided by Prof. Gideon Grogan (York University, UK). This gene was provided in the pPICZα B vector (Invitrogen), which was replaced with the pD902 vector for our work (after performing one silence mutation in the gene to remove SapI restriction site by QuikChange). The resulting construct contains the methanol-inducible AOX1 promotor and the secretion signal of PaDa-I AaeUPO. Hordeum vulgare (barley) oxalate oxidase (HvOXO) gene (GenBank ID: L15737.1) was optimized for Komagataella phaffii (Pichia pastoris) expression and synthesized as an Invitrogen GeneArt Strings DNA fragment (ThermoFisher Scientific). The signal sequence to direct secretion of HvOXO to the cell wall was not included in the DNA synthesized fragment. The mature sequence of HvOXO (GeneBank ID: AAA32959.1) was followed by the recognition site (ENLYFQG) for tobacco etch virus p rotease (TEV) and a 6xHis-tag. The doublestranded DNA fragment was cloned in the pD912 vector. This vector contains the AOX1 promotor and the secretion signal of Saccharomyces cerevisiae mating factor α-1. The optimized sequence of HvOXO-TEV-His-tag, which is not available in the databases, is shown below: > HvOXO-TEV-His-tag  TCTGATCCAGATCCTCTGCAAGATTTCTGTGTCGCTGATTTGGACGGTAAGGCCGTTTCTGTTAACGGTCACACTTGTAAGCCAA  TGTCTGAAGCTGGTGACGACTTCCTGTTCTCCTCAAAGTTGACTAAGGCTGGTAACACCTCCACTCCAAACGGTTCTGCTGTTAC  TGAATTGGACGTTGCCGAATGGCCTGGAACTAACACTTTGGGTGTTTCCATGAACAGAGTCGACTTTGCTCCAGGTGGTACTAAT  CCACCACACATTCATCCAAGAGCTACCGAGATCGGTATGGTCATGAAGGGTGAGTTGTTGGTCGGTATCTTGGGTTCTTTGGACT  CCGGTAACAAGCTGTACTCCAGAGTTGTTAGAGCCGGTGAGACTTTCGTTATCCCAAGAGGTTTGATGCACTTCCAGTTCAACGT  TGGTAAGACCGAGGCCTACATGGTTGTGTCCTTCAACTCTCAAAACCCCGGTATCGTTTTCGTCCCATTGACTTTGTTTGGTTCTG  ACCCACCAATTCCTACTCCAGTTTTGACCAAGGCTTTGAGAGTCGAGGCTGGTGTTGTTGAATTGCTGAAGTCTAAGTTCGCTGG  TGGTTCCGAGAACTTGTACTTTCAAGGTCATCACCACCACCATCACTAA

S1.2. Yeast fermentation
PaDa-I AaeUPO and HvOXO were expressed using K. phaffii as a host (PPS-9010 ATUM) in a 5 L fermentor (B. Braun Biotech International). Plasmid transformation and screening for expression were performed following standard procedures described in ATUM website. The presence of the expression cassette in the yeast genome was confirmed by colony PCR using the Phire Plant Direct PCR master mix (Thermo Scientific) followed by DNA sequencing. To obtain a preculture, a few single colonies were used to inoculate 250 mL of buffered yeast extract peptone dextrose medium (YPD with 200 mM potassium phosphate buffer pH 6.0). The incubation (in a 2.5 L baffled flask) was performed for around 65 h at 30 ºC and 150 rpm (Infors HT Multitron Standard incubator shaker). Before the inoculation with 200 mL preculture, the fermentor contained 3 L basal salts medium (BSM, 1. Initial fermentation was carried out at 30 ºC, 30% pO2 (cascade mode, varying stirring and air flow) and pH 5.0 (controlled using 30% ammonia in water). After glycerol depletion (~20 h), a glycerol feed (5-20% flow, 1.6 mm inner diameter tubing) was performed until the cell density reached approximately 200 g/L. Next, temperature and glycerol feed were gradually decreased to 20 ºC and 0% (~1 h), 12 mL of a 234 mM FeSO4 • 7H2O solution were added to the fermentor and then 43 h of methanol feed were performed (flow, 1.6 mm inner diameter tubing: 2% 6 h, 4% 12 h, 6% 2 h, 7% 2 h, 8% 2 h, 9% 19 h). 1-2 mL antifoam (Struktol J673A) were added each fermentation per day. After fermentation, cells were removed by centrifugation (45 min, 4 ºC, 12,000 x g) and the supernatant (~3 L) was concentrated (~0.250 L) using a tangential flow filtration system (Cogent M, Mettler Toledo, 10 kDa MWCO). Two tablets of protease inhibitor cocktail (cOmplete, EDTA-free, Roche) were added to the concentrated sample.

S1.3. Enzyme purification
Purification of PaDa-I involved both hydrophobic interaction and anion exchange chromatographies using an ÄKTA fast protein liquid chromatography (FPLC) system (Amersham Pharmacia Biotech). First, 40% ammonium sulfate saturation of the sample was achieved by slow addition of solid ammonium sulfate at 4 ºC under continuous stirring (59 g/250 mL). After centrifugation (1 h, 4 ºC, 72,500 x g), the supernatant was subjected to vacuum filtration (0.45 µm PES filter unit, VWR) and applied to three HiTrap Phenyl HP 5 mL connected in series (GE Healthcare). The column equilibration buffer was 20 mM sodium phosphate pH 7.0 containing 1.8 M ammonium sulfate. After loading the sample (5 mL/min), column was washed with five volumes of equilibration buffer and five volumes of the same buffer containing 0.9 M ammonium sulfate. Finally, PaDa-I elution was performed using the same buffer without ammonium sulfate. Next, the resulting PaDa-I sample was concentrated to 15 mL using centrifugal devices (10 kDa MWCO, Pall Macrosep Advance, 4,000 x g, 4 ºC), followed by buffer exchange using a HiPrep 26/10 desalting column (GE Healthcare) equilibrated with 20 mM TrisHCl pH 7.0. Finally, purification of PaDa-I was accomplished by using a RESOURCE Q column (6 mL, GE Healthcare). Mobile phases were 20 mM TrisHCl pH 7.0 and the same buffer with 2 M NaCl. Protein was eluted from the column using a linear salt gradient (0-500 mM NaCl, 70 min, 1 mL/min). Fractions having a Reinheitzahl (RZ) value (A418/A280) of 1.8 -2.2 were pooled, concentrated using centrifugal devices and subjected to buffer exchange using a PD-10 desalting column (Amersham Biosciences) equilibrated with 50 mM potassium phosphate pH 7.0. Purified PaDa-I was flash frozen and stored at -80 ºC.
In the case of the His-tagged HvOXO, gravity-flow bench purification was carried out using Ni-sepharose 6 Fast Flow (GE Healthcare, 3 mL) and standard protocols. 20 mM sodium phosphate pH 7.4 with 0.5 M NaCl was used, which contained 0, 5, and 500 mM imidazole for equilibration, wash and elution, respectively. Before storing the purified samples at -80 ºC, buffer exchange of HvOXO was carried out using a PD-10 desalting column equilibrated with 100 mM citrate-phosphate buffer at pH 4.0.

S1.4. Determination of enzyme concentration
Purified enzyme concentration was determined based on the extinction coefficient at 280 nm: 134.0 mM -1 cm -1 for PaDa-I and 76.4 mM -1 cm -1 for HvOXO. To determine these values according to the Beer-Lambert law, various enzyme dilutions were prepared. Next, their absorbance at 280 nm was measured using a NanoDrop spectrophotometer (ND-1000), while the protein concentration was determined using the Bradford reagent (Sigma-Aldrich).

S1.5. Steady-state kinetics
Steady-state kinetic parameters for HvOXO were determined using potassium oxalate as a substrate, a Varian Cary 300 Bio UV-Visible spectrophotometer and the following PaDa-I-coupled assay. Reactions (100 µL) contained 0.025 µM HvOXO, 0.125 µM PaDa-I, 0.03-200 mM potassium oxalate, 0.07 mM 3-methyl-2-benzothiazolinone hydrazone (MBTH; 1.4 mM stock prepared in water) and 1 mM 3-(dimethylamino)benzoic acid (DMAB; 20 mM stock prepared in methanol) in air-saturated 100 mM citrate-phosphate pH 3.0, 4.0 or 5.0 at 25 ºC. PaDa-I catalyzes the oxidative coupling of MBTH and DMA to form an indamine dye, which exhibits an absorption maximum at 590 nm (Ɛ590 53 mM -1 cm -1 ). [2] 1 µmol of indamine dye is formed per 1 µmol of H2O2 which is produced by HvOXO as a result of the reaction of this enzyme with 1 µmol of potassium oxalate and dioxygen. Potassium oxalate stocks were prepared in 100 mM citratephosphate buffer (20 mM at pH 3.0 and 4.0 and 1 M at pH 5.0). pH of the potassium oxalate stocks was adjusted before adjusting the volume.

S1.6. Melting temperature
Melting temperature values of PaDa-I and HvOXO were determined using a LightCycler® 480 II System (Roche Applied Science) and 384-well PCR plates with qPCR adhesive seals (4titude FrameStar). 4 µL of the enzyme stock prepared in 50 mM potassium phosphate buffer pH 7.0 were mixed with 21 µL 100 mM citrate-phosphate at pH 3.0-8.0. Two 10 µL aliquots of each solution were pipetted into the 384-well plate. Next, 30 nL of 10 mM SYPRO Orange Protein Gel Stain (Sigma-Aldrich, in DMSO) were added using a HP D300 Digital Dispenser to each 10 µL sample. Final concentrations were 0.872 µM PaDa-I (31 µg/mL) or 2.388 µM HvOXO (55 µg/mL), 30 µM SYPRO Orange and 0.3% dimethyl sulfoxide. Fluorescence was measured during enzyme denaturation by increasing the temperature from 20 to 98 °C. [3] Light Cycler 480 SW 1.5.1 software was used to calculate the negative first derivatives (-dF/dt), which reveals melting temperatures as peaks.

S1.8. General indications for the reactions in microplates
All 5 µL reactions were performed in a 384-well low dead volume microplate (Echo qualified, Labcyte) covered with a sealing film (Axygen AxySeal, PCR-SP). Unless stated otherwise, all incubations were performed at 25 °C and 300 rpm in a ThermoMixer with ThermoTop (Eppendorf). 100 mM citrate-phosphate was used as a buffer. A small percentage of an organic solvent (v/v) was used as a cosolvent for substrate solubilization or for investigating enzyme stability as indicated for each experiment. Echo 655 (La bcyte) was used to dispense aqueous solutions or dimethyl sulfoxide. Mosquito HV (sptlabtech) was used to dispense other organic solvents. Reaction product analyses were performed by UPLC-MS or UPLC-QTOF/MS E as described in Section S1.9 and S1.14, respectively. Conversions are relative to the initial substrate concentration, unless stated otherwise, and based on two or three replicates.

S1.9. Work-up and ultrahigh performance liquid chromatography-mass spectrometry (UPLC-MS) for tolmetin-containing reactions
PaDa-I-catalysed 5 µL-scale conversions of tolmetin (final concentration of 0.5, 10 or 100 mM) were stopped by adding 1 µL 3 M HCl and 6 µL dimethyl sulfoxide using an Echo 655 liquid dispenser. After mixing for 5 min at 750 rpm (Heidolph Microtiter Plate Shaker Titramax 1000) and subsequent centrifugation for 1 min at 1500 x g (Eppendorf 5810 R), the supernatant was transferred from the reaction plate (384-well low dead volume microplate, Echo qualified, Labcyte) to the analysis plate (384-well, PP, small volume, deep well, Greiner bio-one) using a Mosquito HV liquid handler. After sealing the plate using a thermal heat sealer (Velocity11´s PlateLoc), 5 µL sample were injected in a Waters Acquity UPLC equipped with a photodiode array (PDA) detector, a 3100 mass spectrometer and an Acquity UPLC BEH C18 column (1.7 µm, 2.1 x 50 mm). To prepare the mobile phases A and B, 4 mL of a basic stock solution (1.625 M NH4HCO3 and 11.269 M NH3 in water) were added to 1 L water (A) and to 1 L 95% acetonitrile in water (B). Method was: 10%B 0.2 min, 10-99%B 1.5 min, and 99-10%B 0.01 min. Flow rate was 1 mL/min.
In the case of 100 µL-scale conversions of 50 mM tolmetin in a 3 mL vial (Fig. 2), reactions were stopped by adding 100 µL 3 M HCl and 600 µL dimethyl sulfoxide to ensure complete solubilization before performing the UPLC-MS analyses described above.

S1.10. Work-up and gas chromatography mass-spectrometry (GC-MS) for ethylbenzene-containing reactions
100 µL-scale conversions of 50 mM ethylbenzene in a 3 mL vial (Fig. 2) were extracted with 300 µL ethyl acetate containing 3 mM phenylacetylene as an internal standard. The organic extracts were dried over MgSO4. Subsequent GC-MS analyses were performed using a J&W HP-5ms GC column (30 m, 0.25 mm, 0.25 µm, Agilent 19091S-433) and He gas as the carrier gas (Agilent Technologies 7890A GC system and 5975C Inert MSD detector). Column temperature was: 50 °C for 3 min, increased to 150 °C at a rate of 15 °C/min, increased to 250 °C at a rate of 60 °C/min and 250°C for 2 min. Injection volume was 1 µL with a split ratio of 30:1. Plots for standards are shown in Fig. S1.

S1.11. Co-solvent stocks preparation for testing their influence on tolmetin conversions
Increasing amounts of various co-solvents (0, 5, 10 and 25%, v/v) were tested in the PaDa-I reactions with either H2O2 or HvOXO and oxalate ( Fig. 5 and S10). Co-solvent stocks contained 55% (v/v) organic solvent in 45 mM citrate-phosphate buffer at pH 4.0 (final concentrations). Citrate-phosphate buffer was prepared by mixing appropriate volumes of 0.1 M citric acid and 0.2 M disodium hydrogen phosphate as indicated in a previous publication. [4] After mixing the buffer with the organic solvent, the pH was adjusted to 4.0 before adjusting the volume with water. All reactions (5 µL, final volume) contained 0.1 µM PaDa -I, 0.5 mM tolmetin and 100 mM citratephosphate buffer pH 4.0. 42 mM tolmetin stock was prepared in dimethyl sulfoxide. Thus, all reactions contained 1% dimethyl sulfoxide in addition to 0-25% of the cosolvent under study. S1.12. Preparation of cross-linked enzyme aggregates which contain two enzymes (combi-CLEA) 0.4 g ammonium sulfate were weighted out in a 1.5-mL tube. Next, 469 µL purified PaDa-I (13.5 µM stock in 50 mM potassium phosphate pH 7.0) and 96 µL purified HvOXO (66.1 µM stock in 100 mM citrate-phosphate pH 4.0) were added to the same tube (final volume of 740 µL). This mixture was incubated at 1000 rpm and 4 °C for 1 h using a ThermoMixer to achieve protein precipitation. Subsequently, 20 µL glutaraldehyde [25% (w/w) stock in water, Sigma-Aldrich] were added and the resultant mixture (0.7% glutaraldehyde, 8 µM PaDa-I, 8 µM HvOXO in 760 µL) was incubated at 1000 rpm and 4 °C for 2 h to achieve crosslinking of the proteins by glutaraldehyde. Immediately after the incubation, the resultant suspension containing the combi-CLEA was added to a 50mg tolmetin solution which was prepared as described in Section S1.13. Subsequently, the combi-CLEA tube was washed with 1 mL 200 mM citrate-phosphate pH 5.0 and the washing was added to the same reaction. Following this protocol, two control samples were prepared: i) control with PaDa-I without HvOXO; and ii) control without both PaDa-I and HvOXO (i.e., ammonium sulfate, glutaraldehyde and buffer). In these controls, the corresponding buffer was added instead of the enzyme.
In order to compare the performance of the combi-CLEA and soluble enzymes, a solution containing 469 µL purified PaDa-I (13.5 µM stock in 50 mM potassium phosphate pH 7.0) and 96 µL purified HvOXO (66.1 µM stock in 100 mM citrate-phosphate pH 4.0) was prepared in a 1.5-mL tube without ammonium sulfate. This mixture was kept at 4 °C while the combi-CLEA sample was being prepared.

S1.13. Preparation of tolmetin-containing reactions on a 50-mg scale and work-up for product isolation and identification
50.1 mg tolmetin (61.4 mg tolmetin sodium salt dihydrate) were weighted out in a 25-mL flask (Duran). Next, 2.607 mL 200 mM citratephosphate pH 5.0 and 3.172 mL potassium oxalate (400 mM stock in 200 mM citrate-phosphate pH 5.0) were added to the flask, which was covered with a rubber stopper (turn-over flange, 30.7 mm, Saint-Gobain Performance Plastics) and incubated for 2 h to ensure maximum solubilization of tolmetin before adding the enzymes (200 rpm, 25 °C, Infors HT Multitron Standard incubator shaker). After the incubation, a solution containing both PaDa-I and HvOXO was added to the flask. Then, 1.196 mL 200 mM citrate-phosphate pH 5.0 were used to wash the enzyme tube and the washing was added to the reaction. Final concentrations in 7.539 mL were: 0.8 µM PaDa-I, 0.8 µM HvOXO, 26 mM tolmetin and 168 mM potassium oxalate. An identical reaction was prepared except for having combi-CLEA (with both PaDa-I and HvOXO) instead of the soluble enzymes, as well as two control reactions. The protocol to prepare the combi-CLEA sample and the controls is detailed in Section S1.12. The pH of the reaction with soluble enzymes and combi-CLEA was determined immediately before stopping them after 96 h of incubation (pH 6.24 and 6.58, respectively) to confirm that there was not a major change in pH respect to the initial one (pH 5.0).

S1.14. Work-up and ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry operating in MS E mode (UPLC-QTOF/MS E ) for drug-containing reactions
PaDa-I-catalysed 5 µL-scale conversions of a diverse drug panel (final concentration of 0.5 mM, Fig. S18-19) were stopped by two sequential 3.5 µL aliquots of acetonitrile using a Mosquito HV liquid handler. After mixing for 15 min at 900 rpm and 25 °C (ThermoMixer with ThermoTop) and subsequent centrifugation for 1 min at 1500 x g (Eppendorf 5810 R), the supernatant was transferred (two sequential 5 µL aliquots) from the reaction plate (384-well low dead volume microplate, Echo qualified, Labcyte) to the analysis plate (384-well, PP, small volume, deep well, Greiner bio-one) using a Mosquito HV liquid handler. Next, the analysis plate was sealed using a thermal heat sealer (Velocity11´s PlateLoc).
Analyses were performed using an Acquity Ultra-Performance Liquid Chromatography (UPLC) system paired with a SYNAPT G2 high definition (HDMS) quadrupole time-of-flight (QTof) mass spectrometer (Waters). Electrospray ionization data were obtained in positive ion mode (ES+). All data were acquired in MS E mode, which allows the exact mass determination for both the precursor and fragment ions in a single analysis by performing the acquisition at low-and high-energy, respectively. Mobile phases were 0.1% formic acid in water (A) and acetonitrile (B). Samples (1-5 µL) were injected into an Acquity UPLC BEHC18 column (130 Å, 1.7 µm, 2.1 mm x 100 mm, Waters). Gradient was: 10-70% B, 6 min. Flow rate was 0.5 mL/min. Leucine enkephalin was used for lock mass at a concentration of 2 ng/µL in 0.1% (v/v) formic acid:acetonitrile (1:1). Data acquisition and processing were performed using M assLynx v4.2 (Waters) and ACD/Spectrus Processor 2020.2.0 (ACD/Labs).  . Arrows indicate the protein band corresponding to either PaDa-I or HvOXO. The molecular weight of the glycosylated HvOXO was determined to be 30-33 kDa, based on the electrophoresis performed under denaturing conditions. This analysis indicates that the carbohydrate content of HvOXO is between 29-36%, since a molecular weight of 21.2 kDa was expected for the non-glycosylated monomeric form. Molecular weight, carbohydrate content and spectral properties of our purified PaDa-I are identical to those described in previous studies. [5] Spectra were recorded for 2 µM PaDa-I and 5 µM HvOXO in 50 mM potassium phosphate pH 7.0. Figure S3. Melting temperature curves of PaDa-I (A) and HvOXO (B) at pH 3.0-8.0. Fluorescence was measured during enzyme denaturation by increasing the temperature from 20 to 98 °C. Light Cycler 480 SW 1.5.1 software was used to calculate the negative first derivatives (-dF/dt), which reveals melting temperatures as peaks. Curves at pH 3.0 were considered an assay artifact and thus the corresponding melting temperature values were not discussed in the manuscript. In the case of HvOXO, we ascribed the first observed increase in fluorescence to hexamer dissociation into inactive subunits. [6] The corresponding Tm values were discussed in the manuscript, while the second observed process was considered not relevant.    [7] The tunnel, which leads from the Mn atom to the surrounding solvent, was calculated using CAVER 3.0.3 PyMol plugin. E58, D60 and E63 lie at the entrance of the tunnel. Interaction between these protonated residues and the oxalate monoanion may facilitate substrate access to the HvOXO active site. [8] The deprotonated carboxylate oxygen atom of oxalate likely binds to the Mn ion, resembling the HvOXO-glycolate complex shown in this figure. [7] Mn atom is bound to the side chains of conserved E95, H90, H137 and H88. When using the stick representation, carbon atoms corresponding to residues and glycolate are colored yellow and green, respectively. Mn atom is depicted as a sphere. Surface mesh of the tunnel volume is colored light green.     In the presence of HvOXO and 8 mM oxalate (instead of H2O2), a 2.6-and 1.5-fold higher conversion yield was observed at 25 and 30 °C, respectively. A significantly reduced conversion was observed using the H2O2-generation system at 40 and 50 °C (9 and 3%). Thus, a temperature of 25 °C is optimal for the PaDa-I reactions containing HvOXO. Similar results were obtained for these experiments at 300 and 900 rpm (despite of having a higher dioxygen transfer rate at 900 rpm), which suggests that the lower solubility of dioxygen at the highest assayed temperatures was not the main reason for the decreased conversions.    6 and 7 in C-E, respectively). The total ion current (TIC, purple) and the ion current resulting from a specified mass (orange) are shown as a function of time. Additional experimental details are described in Section S1.13-S1.14 and Fig. S13. Panel D shows fractions 3-4 which were obtained during the HPLC analyses shown in Fig. S13D (see Fig. S15 for fractions 1-2).     (Table S2). Incubation time was 24 h. (26). Dimerization site on raloxifene was not unequivocally identified. Herein, the major raloxifene dimer produced by CYP3A4, which was characterized by NMR, is shown as an example. In this case, a 1-electron oxidation of the raloxifene 4-hydroxyphenyl moiety took place to form an oxygen-centered radical. Another raloxifene molecule was oxidized on the benzo[b]thiophen-6-ol moiety to form an oxygen-centered radical which converted into the position 7 carbon-centered radical after delocalization. Non-enzymatic coupling of these radicals yielded the dimer. [11] S2.3. Scheme Scheme S1. Catalytic cycle of UPOs. UPOs contain iron protoporphyrin IX (P) in the active site. Ferric resting state (RS) of UPO reacts with H2O2 to form compound-0 (C-0). A glutamate residue (E196 in AaeUPO) facilitates both formation of C-0 and subsequent heterolytic peroxide cleavage to form oxoiron(IV) porphyrin radical cation known as compound-I (C-I). Depending on the UPO, substrate and reaction conditions, C-I participates in either peroxygenative (left) or peroxidative (right) reactions. In the peroxygenative pathway, C-I abstracts one electron and one proton from the substrate (R-H) to generate a substrate radical and the ferryl hydroxide form of UPO (C-II). Next, the substrate radical is hydroxylated by C-II (rebound mechanism). In the peroxidative route, both C-I and deprotonated C-II (in equilibrium with its protonated form) abstract one electron and one proton from the hydroxyl group of a substrate (R-OH). The resulting two substrate radicals are released from the enzyme active site. Modified based on Hofrichter et al. [12] Thus, UPOs combine reaction mechanisms similar to those observed for "classic" peroxidases [13] and P450 enzymes using the "peroxide shunt" or belonging to the peroxygenase family. [14] S2.4. Tables   Table S1. Comparation of H2O2-generation systems for ethylbenzene hydroxylation catalysed by AaeUPO.

Type
Reactants/Devices TONUPO By-product Drawbacks Concept Ref.
First, acetonitrile was tested as a cosolvent for all drugs. Drugs which were not soluble in acetonitrile, were dissolved in tetrahydrofuran or 50% organic solvent. Final drug concentration in the PaDa-I reactions was 0.5 mM with 2.5% cosolvent in all cases. Thus, either 0.125 or 0.250 µL (from the 20 and 10 mM drug stock, respectively) were transferred to the 5 µL reaction (final volume).